RECOMBINANT PSP94 (PROSTATE SECRETORY PROTEIN OF 94 AMINO-ACIDS) DEMONSTRATES SIMILAR LINEAR EPITOPE STRUCTURE AS NATURAL PSP94 PROTEIN

PSP94 has the potential to be a useful diagnostic marker and therapeut ic agent in prostate cancer. Recently, different immunoassay systems f or quantitative analysis of PSP94 in clinical samples have been develo ped, but the epitope structure of PSP94 protein has not been elucidate d. In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein. GST-PSP94 contains antigenic det erminants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the struc ture of natural PSP94 antigen. Since GST-PSP94 was expressed in E. col i and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the prim ary amino acid sequence, rather than conformational structure. This hy pothesis was supported by reciprocal competition in ELISA among natura l, GST-PSP94 fusion protein, and purified recombinant PSP94 protein. T he results demonstrate that the various forms of PSP94 can compete wit h each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity. When natural and rec ombinant PSP94 protein were denatured in vitro with urea and alkali, n o effect on the binding to antibody was found. The epitope activity of natural PSP94 was also shown to be resistant to the treatment of dete rgent and reducing agent. The location of one of the linear epitopes r ecognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequen ces. Competitive ELISA between the N-terminal peptide and PSP94 protei n indicate that both natural and GST-PSP94 have similar immunoactive N -termini. (C) 1996 Wiley-Liss, Inc.